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Cited by 207Mitochondrial Function and PathologyMuscle Physiology and DisordersImmune Response and Inflammation

Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency

Open access
Rodríguez‐Nuevo, Aida, Díaz‐Ramos, Angels, Noguera, Eduard, Díaz‐Sáez, Francisco, Duran, Xavier, Muñoz, Juan Pablo, Romero, Montserrat, Plana, Natàlia +16 more·2018·The EMBO Journal
DOI 10.15252/embj.201796553PMC PMC5978453
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Abstract

Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle-specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF-κB activation, and enhanced expression of pro-inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF-κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell-autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF-κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.

AI snapshot

Written from the paper’s full text.

TL;DR

Muscle-specific loss of Opa1 in mice produces mitochondrial dysfunction, mtDNA stress, and a primary inflammatory response that precedes macrophage infiltration and contributes to growth impairment. The inflammatory NF-κB program depends on mtDNA and TLR9, and anti-inflammatory salicylate treatment partially rescues muscle and growth phenotypes.

Key findings

  • Opa1 ablation in skeletal muscle shortened lifespan, reduced body growth, muscle weight, grip strength, and myofiber cross-sectional area, and altered the GH/IGF1 axis; muscle Fgf21 was strongly induced and circulating Fgf21 was about fourfold higher.
  • Inflammation appeared early: IL-1β labeling was already elevated at 3 weeks, while CD68-positive macrophages were not detected until 13 weeks; NF-κB target genes and plasma IL-1β/IL-6 were increased.
  • Opa1 deficiency caused mitochondrial fragmentation, reduced respiration, increased glycolysis, and mtDNA stress, including roughly 50% lower mtDNA copy number, about half the TFAM protein, fewer nucleoids, and larger nucleoids.
  • The inflammatory response required mtDNA and TLR9: ethidium bromide treatment depleted mtDNA by more than 95% and abolished NF-κB target gene induction, while TLR9 antagonism or TLR9 knockdown reduced NF-κB target genes and IL-6 release; mtDNA co-localized with TLR9 in over 50% of signal.

Methods

  • Mouse models included muscle-specific Opa1 knockout via Opa1-loxP/loxP crossed with myogenin-Cre, tamoxifen-inducible adult muscle Opa1 knockout, cardiotoxin injury with adenoviral Opa1 miRNA, and Opa1/Fgf21 double knockout; treatments included sodium salicylate and chloroquine.
  • Cell studies used C2C12 myoblasts with Opa1 knockdown, ethidium bromide-mediated mtDNA depletion, cGAS or TLR9 knockdown, and ODN2088 TLR9 antagonist treatment.
  • Analyses included histology, immunofluorescence, electron microscopy, mitochondrial respiration, Seahorse extracellular flux, ATP measurement, mtDNA quantification, Western blotting, plasma cytokine/hormone assays, gene expression, and t-tests or ANOVA with post hoc tests at P < 0.05.

Limitations

  • The paper states that mitophagy measurements are difficult in skeletal muscle and that further work is needed to document Opa1’s impact on muscle mitophagy.
  • The paper states that the precise mechanism by which mtDNA interacts with TLR9 remains unclear, that robust demonstration is lacking, and that mitochondria-derived vesicle involvement cannot be excluded.
  • The paper notes that localization of mtDNA or mitochondria to late endosomes could not be concluded because triple staining was lacking; more broadly, the evidence is largely from mouse and C2C12 models, with human inflammatory myopathy relevance proposed rather than directly tested.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.15252/embj.201796553

Cites

65 of 65 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 37 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    CK activity is normalized in salicylate-treated Opa1 KO mice.

    “and also CK activity was normalized (Fig 4D), consistent with reduced muscle necrosis.”
    Quote in the paperSubstantial claim
  • Findingown data

    Muscle-specific Opa1 ablation causes dysfunctional mitochondria.

    “dysfunctional mitochondria”
    Quote in the paperSubstantial claim
  • Findingown data

    Under Opa1 deficiency, blockage or repression of TLR9 prevents NF-kB activation and inflammation.

    “Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-jB activation and inflammation.”
    Quote in the paperSubstantial claim
  • Findingown data

    The effects of Opa1 deficiency on NF-kB target genes and inflammation are absent upon mtDNA depletion.

    “The effects of Opa1 deficiency on the expression NF-jB target genes and inflammation were absent upon mitochondrial DNA depletion.”
    Quote in the paperSubstantial claim
  • Findingown data

    EtBr-treated cells show 95% reduction of mtDNA in Opa1-deficient cells.

    “EtBr-treated cells showed a 95% reduction of mtDNA in Opa1-deficient cells (Fig 6A and Appendix Fig S3A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ethidium bromide treatment depletes mtDNA without affecting other cellular processes relevant to NF-kB target gene expression.
  • May combine two claims. 55
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    Opa1-deficient muscles show increased levels of various mitochondrial proteins, but neither OXPHOS subunits nor Pgc-1a expression.

    “Opa1deficient muscles showed increased levels of various mitochondrial proteins, but neither of OXPHOS subunits nor of Pgc-1a expression (Fig 5B and C and Appendix Fig S2A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Opa1 deficiency causes decreased mitochondrial respiration characterized by enhanced respiratory leak and reduced maximal respiratory capacity.

    “Opa1 deficiency also caused decreased mitochondrial respiration, which was characterized by enhanced respiratory leak and reduced maximal respiratory capacity (Fig 5H)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nucleoid composition analysis reveals reduction in number of nucleoids per cell and enhanced nucleoid size.

    “Nucleoid composition analysis by confocal microscopy revealed a reduction in the number of nucleoids per cell (Fig 5M) and enhanced nucleoid size (Fig 5N).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Chronic sodium salicylate treatment ameliorates muscle alterations and reduces muscle Fgf21 expression.

    “Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 KO mice show enhanced hepatic expression of genes relevant in gluconeogenesis, lipid metabolism, or mitochondrial respiration, and DKO mice show normalized expression.

    “Opa1 KO mice showed enhanced expression of hepatic genes relevant in gluconeogenesis, lipid metabolism, or mitochondrial respiration, and DKO mice showed a normalized expression (Fig 2H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1-deficient muscle sections show substantial necrotic myofibers and regenerating fibers.

    “Histological inspection of Opa1-deficient muscle sections revealed a substantial number of necrotic myofibers, as well as regenerating fibers (centrally located nuclei; Fig 3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Salicylate treatment markedly reduces plasma GH, increases hepatic Ghr, Igf1, Fos, and reduces SOCS3 expression.

    “Salicylate treatment markedly reduced plasma GH (Fig 4E), increased the hepatic expression of Ghr, Igf1, and Fos (Figs 4F and EV3F and G), and reduced SOCS3 expression (Fig 4G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sodium salicylate treatment reduces NF-kB target genes and circulating IL-6 and IL-1b in Opa1 KO mice.

    “Anti-inflammatory efficiency was documented by low expression of NF-jB target genes and by low circulating levels of IL-6 and IL-1b (Fig EV3A-C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 KO mice show decreased hepatic expression of Igf1 and Fos.

    “and decreased expression of its target genes Igf1 and Fos (Fig 2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There are no changes in mitochondrial mass in Opa1-deficient cells, as PORIN, TIM44, SDHA, TOM20 expression is not reduced.

    “There were no changes in mitochondrial mass, as the expression of mitochondrial proteins PORIN, TIM44, SDHA, or TOM20 was not reduced in Opa1-deficient cells (Fig 5G).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Opa1-deficient C2C12 myoblasts show marked increase of NF-kB promoter activity and expression of NF-kB target genes.

    “Opa1 KD myoblasts (Fig EV3C) showed a marked increase of NF-jB promoter activity (Fig 3F) and the expression of NF-jB target genes (Fig 3G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Muscle-specific Opa1 ablation causes muscle inflammation with NF-kB activation and enhanced pro-inflammatory gene expression.

    “muscle inflammation characterized by NF-jB activation, and enhanced expression of pro-inflammatory genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 KO mice show reduced hepatic Ghr expression and increased Socs3 expression.

    “Also, it revealed reduced expression of growth hormone receptor (Ghr) and increased expression of the STAT5 inhibitor Socs3 (Fig 2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Muscle inflammation is an early event during disease progression and occurs before macrophage infiltration.

    “Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Highly positive IL-1b labeling is detected at 3 weeks in Opa1-deficient muscles and maintained with age.

    “Highly positive labeling for IL-1b was already detected at 3 weeks in Opa1-deficient muscles and was maintained with age (Fig 3D and Appendix Fig S1A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 deficiency does not cause necrosis or apoptosis in C2C12 cells.

    “Opa1 deficiency did not cause necrosis (Fig EV2D) or apoptosis (Fig EV2E and F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 ablation causes markedly altered organization of the mitochondrial network and enhanced mitochondrial density in skeletal muscle.

    “Analysis of mitochondrial morphology showed a markedly altered organization of the mitochondrial network and an enhanced mitochondrial density (Fig 5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 KO mice show hepatic alterations including enhanced Socs3, reduced STAT5 phosphorylation, reduced Ghr, and lower GH target genes.

    “We also found hepatic alterations in Opa1 KO mice such as enhanced Socs3 expression, reduced STAT5 phosphorylation, reduced gene expression of growth hormone receptor, and lower expression of growth hormone target genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Rapid recruitment of LC3-II to mitochondrial enriched fractions upon CCCP for 30 min occurs in both control and Opa1-deficient cells, suggesting normal mitophagy induction.

    “A rapid recruitment of LC3-II to mitochondrial enriched fractions was detected upon incubation with CCCP for 30 min in both control and Opa1-deficient cells, suggesting a normal mitophagy induction in Opa1-deficient cells (Fig 7A and Appendix Fig S4C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: CCCP induces mitochondrial depolarization and mitophagy in these cells.

  • May combine two claims.

    Findingown data

    No induction of interferon-stimulated genes nor Ifnb is detected in Opa1-deficient cells.

    “we did not detect induction of interferon-stimulated genes neither Ifnb in Opa1deficient cells (Fig 6E)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 deficiency mitochondrial dysfunction is characterized by mitochondrial fragmentation, reduced mitochondrial respiration, normal or enhanced expression of mitochondrial proteins, and unaltered expression of PGC1a.

    “This is characterized by mitochondrial fragmentation, reduced mitochondrial respiration, normal or enhanced expression of mitochondrial proteins, and unaltered expression of the mitochondrial biogenesis factor PGC1a.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Time-course studies reveal significantly increased p62 and presence of ragged red fibers in muscles of 3-week-old mice, further accumulated with age.

    “Time-course studies revealed significantly increased levels of p62 and presence of ragged red fibers in the muscles of 3-week-old mice, which further accumulated with age (Fig 7H, Appendix Fig S4H and I).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: p62 accumulation reflects impaired autophagic flux in muscle.

  • May combine two claims.

    Findingown data

    DKO mice restore hepatic metabolic gene expression, but growth defects due to Opa1 deficiency are independent of FGF21.

    “Analysis of double knockout mice proved that, although the hepatic expression of metabolic genes was restored, the growth defects due to Opa1 deficiency were independent of FGF21.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 KO mice have low circulating Igf1, high plasma GH, and high Fgf21 in muscle and circulation.

    “The characterization of the Opa1 KO mice growth defects revealed low levels of circulating Igf1, high plasma growth hormone, and high Fgf21 expression in muscle as well as in circulation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Chronic salicylate treatment results in substantial amelioration of muscle mass, body weight, muscle necrosis, and hepatic growth hormone resistance.

    “Furthermore, chronic treatment with salicylate resulted in substantial amelioration of muscle mass, body weight, muscle necrosis, and hepatic growth hormone resistance.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treating Opa1 knockout mice with salicylate reduces inflammation and improves growth.

    “Salicylate treatment reduces inflammation and improves growth in Opa1 KO mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 deficiency is characterized by reduced mtDNA copy number, diminished TFAM protein abundance, reduced number of nucleoids, and increased nucleoid size.

    “Here, we document that Opa1 deficiency was characterized by reduced mtDNA copy number, parallel to diminished TFAM protein abundance, reduced number of nucleoids, and increased nucleoid size.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 deficiency in C2C12 muscle cells causes increased NF-kB transcriptional activity, expression of NF-kB target genes, and production of IL-1b.

    “Opa1 deficiency in C2C12 muscle cells caused increased NF-jB transcriptional activity, expression of NF-jB target genes, and production of IL-1b.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 is necessary for maintaining mitochondrial DNA stability and mitochondrial quality control.

    “In all, we document that Opa1 is required for mtDNA stability and mitochondrial quality control.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Preliminary data suggest that mtDNA and TLR9 may be located together in an intracellular compartment in Opa1-deficient cells.

    “our preliminary data indicate that mtDNA and TLR9 may be present in an intracellular compartment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    mtDNA and TOM20 show complete colocalization in control muscle cells and close apposition in Opa1-deficient cells, suggesting same compartment and that mtDNA is unlikely in the cytosol.

    “Analysis of the distribution of mtDNA and TOM20 (an outer mitochondrial membrane protein) revealed a complete colocalization of both markers in control muscle cells (Appendix Fig S4A and B).The two markers also showed close apposition in Opa1-deficient muscle cells (Appendix Fig S4A and B) suggesting that mtDNA and TOM20 were in the same compartment and that mtDNA is unlikely to be located in the”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Control cells show marked reduction in mitochondrial nucleoids upon 9-h CCCP, while Opa1-deficient cells show no mitophagic response.

    “While control cells responded with a marked reduction in mitochondrial nucleoids upon 9-h incubation with CCCP, Opa1-deficient cells showed no mitophagic response (Fig 7B and C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: CCCP induces mitochondrial depolarization and mitophagy in these cells.

  • May combine two claims.

    Findingown data

    Opa1-depleted muscle shows impaired autophagy flux; chloroquine causes marked LC3-II buildup in control but weak induction in KO.

    “Opa1-depleted muscle also showed an impaired autophagy flux and treatment for 5 days with chloroquine caused a marked buildup of the autophagic protein LC3-II in muscles from control mice, whereas only a weak induction was detectable in Opa1 KO mice (Fig 7G and Appendix Fig S4F).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Chloroquine effectively inhibits lysosomal degradation in muscle and muscle cells.

  • May combine two claims.

    Findingown data

    Control muscles respond to chloroquine by enhanced LC3-II abundance, while Opa1 ablated muscles show only weak non-significant increase.

    “Control muscles responded to chloroquine by enhanced LC3-II abundance (Fig 7D, and Appendix Fig S4D) in keeping with prior data (Sebastian et al, 2016).In contrast, Opa1 ablated muscles only showed a weak non-significant increase in LC3-II (Fig 7D and Appendix Fig S4D).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Sebastian et al, 2016 source

      Not checked in the free run.

    • Rests on an assumption: Chloroquine effectively inhibits lysosomal degradation in muscle and muscle cells.

  • May combine two claims.

    Findingown data

    Opa1 deficiency causes mitochondrial dysfunction both in skeletal muscle and in cultured muscle cells.

    “Here, we report that Opa1 deficiency causes mitochondrial dysfunction both in skeletal muscle and in cultured muscle cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Opa1 depletion causes mtDNA stress in the absence of leakage of mtDNA to the cytosol, with no induction of interferon-stimulated genes, and instead causes NF-kB induction dependent on TLR9 activity.

    “However, here we report that Opa1 depletion causes mtDNA stress, but this occurs in the absence of leakage of mtDNA to the cytosol, and with no induction of interferon-stimulated genes, and instead it causes induction of NF-jB dependent on TLR9 activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Inflammation is initially detected in 3-week-old Opa1 KO mice by enhanced IL-1b abundance and normal CD68 labeling, whereas at 13 weeks CD68 labeling is enhanced, indicating macrophage infiltration.

    “Inflammation was initially detected in 3-week-old Opa1 KO mice by enhanced IL-1b abundance and normal CD68 labeling (a marker of macrophage infiltration), whereas at greater ages (13 weeks of age), CD68 labeling was enhanced, thereby indicating macrophage infiltration.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: CD68 staining specifically marks pro-inflammatory macrophages in this muscle context.

  • May combine two claims.

    Findingown data

    Skeletal muscle-specific Opa1 ablation causes an inflammatory myopathy in mice characterized by enhanced expression of NF-kB target genes and increased circulating levels of IL-1b and IL-6.

    “Thus, we have demonstrated that skeletal muscle-specific Opa1 ablation causes an inflammatory myopathy in mice, which is characterized by enhanced expression of NF-jB target genes, and increased circulating levels of pro-inflammatory cytokines IL-1b and IL-6.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Triple labeling shows exclusion between mtDNA and LAMP1, and apposition of mtDNA and VPS4 signals.

    “Triple labeling of mtDNA, LAMP1, and VPS4 in control and Opa1-deficient cells showed exclusion localization between mtDNA and LAMP1, and apposition of mtDNA and VPS4 signals (Appendix Fig S6C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    TLR9 interaction with mtDNA activates NF-kB signaling and increases pro-inflammatory cytokine expression.

    “Interaction of TLR9 with mtDNA activates the nuclear factor kappa B (NF-jB) signaling and increases the expression of other pro-inflammatory cytokines such as tumor necrosis factor-a (TNF-a), interleukin (IL)-6, IL-1b (Julian et al, 2013;Yu & Bennett, 2014;Zhang et al, 2014).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Julian et al, 2013 source

      Not checked in the free run.

    • cites Yu & Bennett, 2014 source

      Not checked in the free run.

    • cites Zhang et al, 2014 source

      Not checked in the free run.

  • May combine two claims.

    Premisereasoning

    Patients with OPA1 mutations show multiple mtDNA deletions in muscles, suggesting a role for OPA1 in mtDNA stability.

    “Interestingly, these patients show multiple mitochondrial DNA (mtDNA) deletions in muscles, which suggests a role of OPA1 on mtDNA stability.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Opa1-deficient muscle undergoes a primary inflammatory process characterized by NF-kB activation, followed by necrosis and then macrophage infiltration.

    “In all, our data indicate that Opa1-deficient muscle undergoes a primary inflammatory process characterized by NF-jB activation, followed by necrosis and then macrophage infiltration.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation and inflammation, which contributes to enhanced Fgf21 expression and growth impairment.

    “Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mitochondrial fusion in mammalian cells is regulated by Mfn1, Mfn2, and Opa1.

    “Mitochondrial fusion in mammalian cells is regulated by the proteins mitofusin 1 and mitofusin 2 (Mfn1 and Mfn2) and optic atrophy 1 (Opa1).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Twig et al, 2008 source

      Not checked in the free run.

    • cites Liesa et al, 2009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    OPA1 mutations cause ADOA plus syndrome, which includes severe myopathy.

    “In addition to dominant optic atrophy, OPA1 mutations also cause a multi-systemic disorder called ADOA plus syndrome, which results in severe myopathy (Amati-Bonneau et al, 2008;Hudson et al, 2008;Zeviani, 2008).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Amati-Bonneau et al, 2008

      Not checked in the free run.

    • cites Hudson et al, 2008 source

      Not checked in the free run.

    • cites Zeviani, 2008 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cytosolic mtDNA activates cGAS and promotes STING-IRF3-dependent signaling.

    “Thus, cytosolic mtDNA activates the DNA sensor cGAS and promotes STING-IRF3-dependent signaling (Rongvaux et al, 2014;White et al, 2014;West et al, 2015).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Rongvaux et al, 2014 source

      Not checked in the free run.

    • cites White et al, 2014 source

      Not checked in the free run.

    • cites West et al, 2015 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Opa1 repression-induced NF-kB activation is cell-autonomous and independent of cell death.

    “thereby revealing that the activation is a cell-autonomous process and independent of cell death.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Fgf21 mediates metabolic alterations in liver from Opa1 KO mice, but is not involved in their reduced growth.

    “In all, these data indicate that Fgf21 mediates the metabolic alterations occurring in liver from Opa1 KO mice, but it is not involved in the reduced growth of Opa1 KO mice.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Opa1 is involved in inner mitochondrial membrane fusion and cristae morphogenesis.

    “Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Olichon et al, 2003 source

      Not checked in the free run.

    • cites Cipolat et al, 2004 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Fgf21 causes resistance to growth hormone.

    “Given the observation that Fgf21 causes resistance to GH (Inagaki et al, 2008)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Inagaki et al, 2008 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    mtDNA activates immunity through TLR9 and cGAS pathways.

    “MtDNA has been reported to activate immunity through two distinct pathways, namely Toll-like receptor 9 (TLR9; Zhang et al, 2010;Oka et al, 2012;Liu et al, 2015), and cGAS activation (White et al, 2014;West et al, 2015).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Zhang et al, 2010 source

      Not checked in the free run.

    • cites Oka et al, 2012 source

      Not checked in the free run.

    • cites White et al, 2014 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Opa1 deficiency in muscle causes global growth defects in conjunction with an altered Igf1 pathway.

    “These data are consistent with the view that Opa1 deficiency in muscle causes global growth defects in conjunction with an altered Igf1 pathway (Wong et al, 2016).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Wong et al, 2016 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Overexpression of IL-6 causes reduced circulating Igf1 and reduced growth, rescued by anti-IL-6 antibodies.

    “The use of mouse models has documented that overexpression of IL-6 causes reduced circulating levels of Igf1 and reduced growth which is rescued by blocking anti-IL-6 antibodies (De Benedetti et al, 1997).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites De Benedetti et al, 1997 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Opa1 deficiency causes muscle inflammation independent of muscle mass loss, occurs before macrophage infiltration, and is mediated via NF-kB activation.

    “On the basis of these data, we conclude that Opa1 deficiency causes muscle inflammation which is independent of muscle mass loss, that occurs before macrophage infiltration, and that it is mediated via activation of NF-jB.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Opa1 deficiency induces mtDNA stress including mtDNA loss and reduced nucleoid number.

    “Our data support the existence of mtDNA stress induced by Opa1 deficiency, which includes mtDNA loss and reduced nucleoids number.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Opa1 deficiency induces low mtDNA copy number and nucleoid abundance in human fibroblasts, suggesting alterations not restricted to muscle cells.

    “Opa1 deficiency has been reported to induce low mtDNA copy number and nucleoids abundance in human fibroblasts, which suggests that those alterations are not just restricted to muscle cells (Liao et al, 2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Liao et al, 2017 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Anti-inflammatory treatment normalizes FGF21 expression and circulation, suggesting enhancement of the mitokine is downstream of inflammation.

    “Interestingly, anti-inflammatory treatment also normalized FGF21 expression and its circulation levels, suggesting that the enhancement of the mitokine is downstream of inflammation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    High plasma FGF21 reduces growth hormone signaling, resulting in a marked growth defect.

    “Given that high plasma concentrations of FGF21 have been observed to reduce growth hormone signaling, resulting in a marked growth defect (Inagaki et al, 2008)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Inagaki et al, 2008 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    TNF-a, IL-1b, and IL-6 induce hepatic growth hormone resistance in mice.

    “In addition, TNF-a, IL-1b, and IL-6 induce hepatic growth hormone resistance in mice (Zhao et al, 2014).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Zhao et al, 2014 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Inflammation regulates growth.

    “Inflammation has been reported to regulate growth (O' Connor et al, 2008).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites O' Connor et al, 2008 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Growth inhibition is commonly observed in children with chronic inflammatory diseases such as juvenile idiopathic arthritis, chronic kidney disease, or chronic inflammatory bowel disease.

    “Growth inhibition is commonly observed in children with chronic inflammatory diseases such as juvenile idiopathic arthritis, chronic kidney disease, or chronic inflammatory bowel disease (Wong et al, 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wong et al, 2016 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Specific ablation of Opa1 in skeletal muscle results in whole body growth impairment, systemic inflammation, and premature death.

    “In this study, we show that specific ablation of the mitochondrial fusion protein Opa1 in skeletal muscle results in whole body growth impairment, systemic inflammation, and premature death.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In these patients, circulating concentrations of pro-inflammatory cytokines such as IL-1b and IL-6 are elevated.

    “In these patients, circulating concentrations of pro-inflammatory cytokines such as IL-1b and IL-6 are elevated (Kutukculer et al, 1998;Ji et al, 2002).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kutukculer et al, 1998 source

      Not checked in the free run.

    • cites Ji et al, 2002

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Autophagy malfunction causes mtDNA-mediated inflammation.

    “Based on data indicating that autophagy malfunction caused mtDNA-mediated inflammation (Nakahira et al, 2011;Oka et al, 2012)”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Nakahira et al, 2011 source

      Not checked in the free run.

    • cites Oka et al, 2012 source

      Not checked in the free run.

64 of 64

  • The paper relies on this without support.

    CD68 staining specifically marks pro-inflammatory macrophages in this muscle context.

    “pro-inflammatory macrophages (stained for CD68)”
    “a marker of macrophage infiltration”

  • The paper relies on this without support.

    IL-1b labeling indicates inflammation in muscle.

    “Highly positive labeling for IL-1b was already detected at 3 weeks in Opa1-deficient muscles”

  • The paper relies on this without support.

    Ethidium bromide treatment depletes mtDNA without affecting other cellular processes relevant to NF-kB target gene expression.

    “We depleted mtDNA in C2C12 muscle cells by chronic treatment with ethidium bromide (EtBr).”

  • The paper relies on this without support.

    ODN2088 specifically antagonizes TLR9 in these cells.

    “cells were treated with the TLR9 antagonist ligand ODN2088 or with the corresponding negative control.”

  • The paper relies on this without support.

    Chloroquine effectively inhibits lysosomal degradation in muscle and muscle cells.

    “treatment for 5 days with chloroquine caused a marked buildup of the autophagic protein LC3-II in muscles from control mice”

  • The paper relies on this without support.

    CCCP induces mitochondrial depolarization and mitophagy in these cells.

    “Mitochondrial autophagy was analyzed upon treatment of control of Opa1-deficient muscle cells with the depolarizing agent CCCP.”

  • The paper relies on this without support.

    p62 accumulation reflects impaired autophagic flux in muscle.

    “Time-course studies revealed significantly increased levels of p62”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Inhibition of OPA1 in muscle results in a similar but more severe phenotype than skeletal muscle MFN1 and MFN2 ablation.

    “Inhibition of the IMM profusion factor, OPA1, in muscle resulted in a similar but more severe phenotype than skeletal muscle MFN1 and MFN2 ablation [187, 192, 193].”

    From The connection between the dynamic remodeling of the mitochondrial network and the regulation of muscle mass (2021) · cites this paper as [192]

  • When OPA1 was deleted during fetal development, animals died a few days after birth.

    “When OPA1 was deleted during fetal development, animals died a few days after birth.”

    From The connection between the dynamic remodeling of the mitochondrial network and the regulation of muscle mass (2021) · cites this paper as [192]

  • Deletion of OPA1 in adult mice (3-5 months of age) results in a decrease of mtDNA, reduced respiratory complexes and supercomplexes content, and reduced complex activity, respiration, and mitochondrial membrane potential.

    “Deletion of OPA1 in adult mice (3–5 months of age) resulted in a decrease of mtDNA, reduced respiratory complexes and supercomplexes content, and complex activity, respiration, and mitochondrial membrane potential.”

    From The connection between the dynamic remodeling of the mitochondrial network and the regulation of muscle mass (2021) · cites this paper as [192]

  • Muscle-dependent systemic release of FGF21 increases with starvation, endoplasmic reticulum stress, mitochondrial dysfunction, obesity, mitochondrial myopathies, and aging.

    “In contrast, muscle‐dependent systemic release of FGF21 increases with starvation, endoplasmic reticulum stress, mitochondrial dysfunction, obesity, mitochondrial myopathies, and aging.6, 13, 14, 15, 16, 17, 18, 19”

    From Fibroblast growth factor 21 controls mitophagy and muscle mass (2019) · cites this paper as [16]

  • In a muscle-specific OPA1 knockout animal model, the contribution of skeletal muscle to circulating FGF21 is predominant.

    “Moreover, we and others recently demonstrated that in a muscle‐specific OPA1 knockout animal model, characterized by mitochondrial dysfunction and by extensive muscle loss, the contribution of skeletal muscle to circulating FGF21 was predominant.15, 16, 18”

    From Fibroblast growth factor 21 controls mitophagy and muscle mass (2019) · cites this paper as [16]

Cites

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Related

Authors
Rodríguez‐Nuevo, A., Díaz‐Ramos, A., Noguera, E., Díaz‐Sáez, F., Duran, X., Muñoz, J. P., Romero, M., Plana, N., Sebastián, D., Tezze, C., Romanello, V., Ribas, F., Seco, J., Planet, E., Doctrow, S. R., González, J., Borràs, M., Liesa, M., Palacín, M., Vendrell, J., Villarroya, F., Sandri, M., Shirihai, O., & Zorzano, A.
Affiliations
  • Instituto de Salud Carlos III (ES)
  • Universitat de Barcelona (ES)
  • Veneto Institute of Molecular Medicine (IT)
  • Institute for Research in Biomedicine (ES)
  • Boston University (US)
  • Parc Científic de Barcelona (ES)
  • University of California, Los Angeles (US)
ORCID
Venue
The EMBO Journal
Publisher
Springer Science and Business Media LLC
Published
2018-04-06
Volume
37
Issue
10
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
DOI
10.15252/embj.201796553
PMC
PMC5978453
OpenAlex
W2796990937
Semantic Scholar
f7306933c3e1fbdf751d9e98b5e032c8112b682d
Cited by
207
References
65
Citations by year
2026: 11 · 2025: 24 · 2024: 26 · 2023: 32 · 2022: 24 · 2021: 40 · 2020: 30 · 2019: 13 · 2018: 6
FWCI
7.87
Type
journal article
Language
en
Open access
Licence
Locations
  • The EMBO Journal (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Padua Research Archive (University of Padova) (submittedVersion, other-oa)
Funders
  • “Ministerio de Educación y Cultura,” Spain
  • Fundación Isabel Gemio
  • FEDER y ASEM, Instituto de Salud Carlos III (PIE14/00045)
  • INTERREG IV‐B‐SUDOE‐FEDER (SOE1/P1/E178)
  • Ministerio de Economía y Competitividad (SAF2013‐40987R, SAF2016‐75246R)
  • Generalitat de Catalunya (2014SGR48)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
OPA1, mitochondrial DNA, TLR9, muscle inflammation, skeletal muscle, NF-κB activation, FGF21, mitochondrial fusion, muscle fiber size, growth retardation, sodium salicylate
MeSH terms
Animals, Cells, Cultured, DNA, Mitochondrial, Female, Inflammation, Male, Muscular Diseases, Necrosis, Regeneration, Cytokines, Apoptosis, Mice, Knockout, Muscle, Skeletal, GTP Phosphohydrolases, Toll-Like Receptor 9

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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